The objective of this research proposal is to reversibly modulate protein-ligand affinity by a thermally-triggered molecular switch attached to the protein scaffold. A polypentapeptide of elastin (1Val-2Pro-3Gly-4Val-5Gly)n, which displays an inverse temperature transition, will be incorporated into tendamistat, a tight-binding protein inhibitor of a-amylase, in the vicinity of its a-amylase binding site. Genetic circular permutation will be used to synthesize and attach the elastin peptide to the tendamistat scaffolding at the gene level. To our knowledge, this is the first use of genetic circular permutation to engineering a peptide switch in a protein to modulate its function. The advantage of genetic circular permutation over chemically conjugating the peptide to the protein is that it eliminates the need for a subsequent conjugation step and provides control over the site of incorporation as well as 100% yield of the desired product by heterologous protein expression. We hypothesize that the thermally-triggered, intramolecular, disorder-order structural transition of the elastin polypeptide in the vicinity of tendamistate's a-amylase binding site is a switch that can perturb the microenvironment, leading to reversible modulation of protein function. The reversible modulation of functional activity of the circularly-permuted tendamistat/elastin hybrids ("permustat/elastin") will be investigated by temperature-dependent a-amylase inhibition assays. While preliminary results demonstrating the feasibility of circularly permuting tendamistat are presented, affinity modulation of ligand binding needs to be clearly demonstrated to establish the feasibility of this approach. Funding through the R21 mechanism will allow us to test the validity of our protein engineering strategy and functional hypotheses. The proposed research will provide fundamental insight into the mechanism of binding and control exerted by "smart" - environmentally sensitive-modulators of protein function. The "smart" affinity switches of protein function, proposed here, will be exploited in the development of regenerable biomedical sensors and hyperthermic drug delivery vehicles.